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DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamin
DOI: Reference:
http://dx.doi.org/10.1016/j.comptc.2015.11.016 COMPTC 1997
To appear in:
Computational & Theoretical Chemistry
Received Date: Revised Date: Accepted Date:
24 September 2015 16 November 2015 17 November 2015
Please cite this article as: A.G. Papadopoulos, N. Nenadis, M.P. Sigalas, DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamin, Computational & Theoretical Chemistry (2015), doi: http://dx.doi.org/10.1016/j.comptc.2015.11.016
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DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamin Anastasios G. Papadopoulosa, Nikolaos Nenadisb,*, Michael P. Sigalasa,**
a
Aristotle University of Thessaloniki, School of Chemistry, Laboratory of Applied
Quantum Chemistry, Thessaloniki 54124, Greece b
Aristotle University of Thessaloniki, School of Chemistry, Laboratory of Food
Chemistry and TEchnology, Thessaloniki 54124, Greece
**Corresponding author. Tel.: +30 2310 997815. E-mail address: [email protected] (M.P. Sigalas). * E-mail address: [email protected]
Abstract DFT calculation of various molecular descriptors was carried out in order to examine the radical scavenging properties of sesame oil lignans and some selected metabolites of sesamin formed in vivo. The major sesame oil lignans, namely sesamin and sesamolin may present antioxidant activity through hydrogen atom transfer, though lacking a phenolic group. This can be achieved via the contribution of allylic hydrogen atoms according to C-H bond dissociation enthalpy (BDE) values. Even so, the predicted activity is not higher than that of some phenolic derivatives (e.g. pinoresinol) or metabolites bearing catechol moieties. The contribution of nonphenolic hydrogen atoms may result in the formation of planar or semi-planar conjugated compounds. Some of the tested lignans (e.g. sesaminol, sesamolinol) may be efficient electron donors comparable to active flavonoids according to ionization potential (IP) values. Neutral sesamol can be an efficient electron donor rather than hydrogen atom donor according to IP and net electrophilicity values. Only computation in water could partially justify literature experimental findings on the higher reactivity of sesamol over that of sesamin. The metabolites of sesamin, bearing one or two catechol groups were found as the most efficient hydrogen atom donors and most prone to ionization. Therefore, they could be capable to act efficiently via hydrogen atom transfer (HAT) or sequential proton loss electron transfer (SPLET) in real systems as shown by gas-phase and calculations in solvents (benzene, water). Thus, these metabolites may account for the high in vivo antioxidant activity of sesamin. Examination of the succeeding products of metabolism indicated the progressive loss of radical scavenging efficiency which was predicted to be negligible in the compounds excreted from the body of mammals. Keywords: DFT; sesame oil; lignans; sesamin; pinoresinol; radical scavenging
1. Introduction Sesame seed is a pool of phytochemicals with significant nutritional importance and health-promoting properties [1]. Among these compounds, a set of furanolignans, namely sesamin, sesamolin and some minor ones (sesamol, sesaminol, sesamolinol, pinoresinol, matairesinol, lariciresinol and episesamin) have a broad spectrum of biological properties, including the suppression of lipid peroxidation [2,3]. The fat soluble ones, found in the sesame oil have been reported to contribute to the exceptional antioxidant activity of the oil, acting even synergistically with tocopherols and Maillard reaction products [3]. Due to their biological importance there is a raising interest in isolating lignans in high purity for medicinal applications [2] and even in the development of strategies aiming to obtain varieties with higher lignan content [1]. In this way a crop of higher nutritional and functional value may be obtained, which can produce edible oil with higher shelf-life and added value. Despite the fact that sesame lignans are of high importance for the food and pharmaceutical sector, there is limited theoretical information concerning their electronic properties, and particularly those related to their radical scavenging activity. The latter is possibly due to the fact that major sesame lignans do not bear any hydroxyl group, whereas on the basis of some in vitro experiments are considered rather poor antioxidants, although there are contradictory findings [3]. Thus, information available regards pinoresinol [4], a derivative of sesamin hydrolysis during oil production, or sesamol [5, 6] deriving from sesamolin under the same conditions, both of which bear a hydroxyl group attached to the aromatic ring. Other theoretical studies, on e.g. pinoresinol are related to conformation analysis [7]. Taking into account the above, B3LYP density functional theory calculations were used to investigate the structure-radical scavenging activity of major and minor
sesame lignans (Fig. 1A) Furthermore, the study has been extended to a set of metabolites of sesamin which can be formed in the liver of mammals, together with those forms eventually excreted in their urine (Fig. 1B). Some of these metabolites due to the presence of catechol moieties have been considered as the active forms responsible for the in vivo antioxidant effect of lignans [3].
Fig. 1 Sesame lignans and sesamol (A), and its in vivo metabolites (B) under study as cited in ref [2].
A series of molecular descriptors (bond dissociation enthalpy, BDE; ionization potential, IP; proton dissociation enthalpy, PDE; proton affinity, PA; electron transfer energy, ETE; net electrophilicity, Δɷ±) characterizing the hydrogen atom or electron donating efficiency of the selected compounds were computed [8-11]. Calculations were first carried out in the gas-phase as good primary indices for radical scavenging [12, 13]. Solute–solvent interactions were also considered using theoretical models
approximating the activity of test compounds in lipid or in aqueous media. With the current study we expect to add to the characterization of the antioxidant properties of the respective compounds and stimulate the interest for further studies and exploitation by the food and pharmaceutical industries.
2. Computational details
All calculations were performed by the Gaussian 03W ver. 6.0 set of programs [14]. The hybrid DFT method with Becke’s three-parameter functional [15] and the nonlocal correlation provided by the Lee, Yang, Parr expression [16] (B3LYP) was used for geometry optimization using the 6-311G basis set (unrestricted B3LYP for the optimization of the resulting radicals). Frequency calculations were performed at the same level of theory to characterize the nature of the stationary points. Singlepoint electronic energies were then obtained using the 6-311++G (2d,2p) basis set. Employing the electronic energies (298 K) at 6-311++G (2d,2p) and thermal contributions to enthalpy obtained at 6-311G, the bond dissociation enthalpy (BDE) values that characterize hydrogen atom donating activity (HAT) were determined according to the equation: BDE = Hr + Hh – Hp, where Hr is the enthalpy of the radical generated by H-atom abstraction, Hh is the enthalpy of the H-atom (−0.499897 hartree at this level of theory), and Hp is the enthalpy of the parent molecule. In the examination of sequential loss of hydrogen atoms, BDE was calculated taking into account the appropriate species. The ionization potential (IP) values characterizing single electron (SET) transfer efficiency were determined according to the equation: IP = Ecr − Ep, where p and cr stand for the parent molecule and the corresponding cation radical generated after electron transfer. Proton affinity
(PA) values defining the first step of sequential proton loss electron transfer (SPLET) mechanism were calculated according to the formula: PA = Ha + Hpr − Hp, where Ha is the enthalpy of the anion generated after deprotonation, Hpr is the enthalpy of the proton (0.00236 hartree), and Hp is the enthalpy of the parent molecule. The electron transfer energy (ETE) or otherwise ionization potential values of the anions were obtained via the equation: ETE = Er – Ea, where r and a are referred to the phenoxy radical and the anion, respectively. No spin contamination was found for radicals, with the values being about 0.750 in all cases. The net electrophilicity Δɷ± was computed as described by [8]. The solvent effect was considered by employing the self-consistent reaction field method with the integral equation formulation-polarized continuum model (IEF-PCM) to do a single point calculation on the B3LYP/6311++G (2d,2p) according to the approach followed in past studies on phenolic antioxidants [4, 17]. Such an approach was adopted considering the number/size of the test compounds and the numerous calculations to be carried out. Even so, before applying the respective methodology the in solvent values for each descriptor were computed for simple phenol without or after structure optimization in solvent. The differences were less than 0.8 %.
3. Results and discussion 3.1. Conformation analysis To compute the appropriate values of the selected molecular descriptors that may characterize the antioxidant activity of the compounds, the conformational space of the molecules under investigation has been explored in order to locate the structure of the global minimum among all the possible conformers. For each molecule a series of
conformations being minima with energies close to this of the global minimum of the potential energy surface have been located. Thus, 2 local minima in an energy window of 11.4 kcal/mol above the global minimum have been found for sesamin, 1 in 1.5 kcal/mol for epipesamin, 7 in 5.3 kcal/mol for sesamolin, 12 in 14.5 kcal/mol for sesaminol, 4 in 2.9 kcal/mol for sesamolinol, 5 in 13.7 kcal/mol for pinoresinol, 1 in 0.3 kcal/mol for sesamol, 9 in 7.5 kcal/mol for M3, 5 in 7.9 kcal/mol for M4, 11 in 12.8 kcal/mol for M6, 2 in 10.6 kcal/mol for M10, 1 in 5.3 kcal/mol for M11, 2 in 0.3 kcal/mol for M1 and 1 in 0.2 kcal/mol for M2. The optimized structure of the global minimum for all the lignans, sesamol and sesamin metabolites are presented in Fig. 2. In all molecules bearing dioxabicyclooctane system, the two five-membered rings are cis-fused and furan ring adopts a half-chair conformation. There is a satisfactory agreement between the calculated structural parameters and those derived from X-ray studies for sesamin [18-20], episesamin [20-23], sesaminol [24], pinoresinol [25] and lariciresinol [25]. The local minima found have the same conformation of the dioxabicyclooctane system and differ in the tortion angle defining the orientation of the benzodioxole ring.
Fig.2. Optimized structures of the molecules studied at the B3LYP/6-311G level (hydrogen atoms not shown for clarity and are given as supplementary material) 3.2. Hydrogen atom transfer In the process of radical scavenging activity, the number and the relative position of hydroxyl groups in phenolic compounds are important [26]. However, on the basis of quantum chemical calculations it has been suggested that allylic hydrogen atoms may also participate to the scavenging of free radicals. In some cases, namely in catechins, such hydrogen atoms may compete with those of the hydroxyl groups [27], whereas in other cases they may follow in donation as it has been predicted for dihydroxy-caffeic acid [26] or some synthetic isochroman derivatives of hydroxytyrosol [13]. A non phenolic hydrogen atom, other than allylic, has recently been proposed to be of significant importance for the radical scavenging of oleuropein according to DFT calculations [28]. Due to the lack of hydroxyl group in the major sesame lignans, namely sesamin and sesamolin, inevitably calculations were focused on the possible donation of allylic hydrogen atoms. Thus, it was found that in sesamin the allylic hydrogen atom at C-4 can be abstracted considering the difference in BDE value (ΔBDE= -7.9 kcal/mol) calculated relatively to that of the hydroxyl group of phenol (BDE= 84.4 kcal/mol). Phenol is expected to be inactive, unless the free radical is very drastic (e.g. ●
OH), and it may serve as a reference compound [9]. Furthermore, the BDE value for
the allylic hydrogen is only 1.5 kcal/mol higher than that computed for catechol (75.2 kcal/mol), an essential structural feature for adequate antioxidant activity via HAT. Trying to examine further the antioxidant potential of sesamin when the HAT is dominant, we have explored a possible mechanism depicted in Fig. 3. For each step
the energetically favored abstraction was located by optimizing radicals derived by all the possible hydrogen donated.
Fig. 3. Proposed scavenging of free radicals by sesamin via sequential hydrogen atom donation
It seems that after the allylic atom abstraction, the donation of a hydrogen atom from a vicinal carbon at C-5 is feasible so that the molecule to become stable via the formation of a double bond. The corresponding value is too low in comparison to that of phenol (ΔBDE= -34.0 kcal/mol), which is rather expected considering that the derived molecule is more stable. The value is of the same magnitude to that calculated for other C-H bonds with low BDE which can participate in reaction eventually leading to more stable structure [10, 29]. Considering the symmetry of the compound a second allylic hydrogen atom seems possible for abstraction at C-8 (ΔBDE= -6.3 kcal/mol), followed by a second hydrogen atom abstraction from the neighboring carbon C-1 (ΔBDE= -45.0 kcal/mol), resulting in a second double bond formation. The BDE value at C-8’ was found considerably higher than that of phenol (ΔBDE= +5.1 kcal/mol). Thus, it is not expected to add to the antioxidant potency of the compound, although studies calculating energies of activation or rate constants with individual radical can be useful to clarify this [6]. Consequently sesamin seems to be able to donate four hydrogen atoms with a sum of the four ΔΒDE values equal to -
93.4 kcal/mol. The ΔΒDEsum was used as a measure of the total relative to phenol enthalpy resulting from the contribution of all hydrogen atoms that could be donated by the test compound. The proposed mechanism affects the molecular geometry, as the formation of double bonds results in a conjugated planar molecule, contrary to the mother compound (sesamin). Similar findings to those evidenced for sesamin were observed for sesamolin regarding the part of the molecule that bears an allylic hydrogen. Thus, ΔBDE values calculated for the abstraction of the corresponding hydrogen at C-8 and the subsequent formation of a double bond after abstraction at C-1 were -7.8 and -31.3 kcal/mol respectively. Nevertheless, in the other part of the molecule, the introduction of an oxygen group between the furan and the aromatic ring changes the electronic phenomena. Therefore, the C-H BDE value at C-4 became higher than that computed for phenol (ΔBDE +7.1 kcal/mol). Consequently, sesamolin is expected to be less efficient than sesamin, considering that it may donate only two hydrogen atoms (ΔBDEsum= -39.0 kcal/mol). As already mentioned for sesamin, the formation of a double bond is expected to affect the geometry of the molecule making it partially planar. Conversion of sesamin to its geometrical isomer (episesamin) during refining of the oil is not expected to affect significantly the BDE values. This is likely taking into account that the calculated values are affected by the local phenomena rather than the whole structure [30]. Indeed the ΔBDEsum= -92.0 kcal/mol, that is only 1.4 kcal/mol higher than that of sesamin. Steric effects on the other hand may influence the interaction with the free radicals and therefore the radical scavenging efficiency. Molecules that derive from the major lignans during the oil production and bear a phenolic group, namely sesaminol, pinoresinol and sesamolinol were then
examined. In the case of sesaminol, the abstraction of the allylic hydrogen atom at C-6' followed by formation of a double bond was preferable than that of the phenolic hydrogen atom. The obtained values were almost comparable to those of sesamin (ΔBDEsum= -93. 0 kcal/mol). In addition, the hydrogen atom donation from the phenolic group which was not favored as a primary site of donation (84.4 kcal/mol), was now predicted to be easily prone to abstraction (ΔBDE= -9.4 kcal/mol). The latter is probably due to the extended conjugation formed in the compound which stabilizes more effectively the derived phenoxy radical. This is illustrated clearly in Fig. 4, where spin delocalization takes place not only in the aromatic ring, as it would be in case the phenolic hydrogen atom is abstracted first, but also through both the fused five- member rings. As a consequence the BDE value significantly becomes lower. Thus, sesaminol is expected to be more efficient than sesamin, considering that the ΔBDEsum for the five hydrogen atoms was -102.5 kcal/mol. In pinoresinol, the same tendency was observed, leading to a planar and fully conjugated molecule. After its formation, a phenolic hydrogen atoms could further be abstracted easily at C-4' (ΔBDE= -10.5 kcal/mol). This is probably due to the extended conjugation of the compound (Fig. 4), as previously proposed for sesaminol.
Fig. 4. Spin density distribution of sesaminol and pinoresinol phenoxy radicals
However, donation of the second one may be rather not feasible (ΔBDE= +0.9 kcal/mol), at least with not very reactive free radicals, despite the fact that the quinone (paired-electron system) should be more stable than a phenoxy radical (unpaired electron system). The respective approach indicated that pinoresinol should be much more efficient radical scavenger than predicted in the past where calculations only considered the BDE value of the phenolic groups [4]. Furthermore, it should be more efficient than sesamin (ΔBDEsum= -104.3 kcal/mol) in accordance with experimental findings [31]. Contrary to what observed in the above two compounds, the primary hydrogen atom for donation in sesamolinol was found to be this of the phenolic group at C-4" (ΔBDE= -7.4 kcal/mol). This is rather surprising considering that sequential hydrogen atom donation starting from the allylic hydrogen atom donation from C-4 was still feasible (ΔBDE= -5.2 kcal/mol), despite the fact that an unstable system bearing two un-paired electrons would be formed in this way. Even so, a rapid hydrogen atom donation from the vicinal carbon at C-5 could follow (ΔBDE= -34.4 kcal/mol) leading to a more stable phenoxy radical. Similarly to sesamolin, the hydrogen atom at C-8 seems not feasible for donation (ΔBDE= +8.3 kcal/mol). Nevertheless, sesamolinol is expected to be more potent than sesamolin due to the presence of the phenol group (ΔBDE= -47.1 kcal/mol). Sesamol is the simplest compound deriving from sesamolin hydrolysis during roasting. It is considered essentially an efficient radical scavenger using various in vitro assays as summarized by Wan et al. [3]. As a matter of fact it has been found more efficient in the scavenging of DPPH● and superoxide anion than sesamin and sesamolin, and of comparable activity to that of pinoresinol [31]. Such an activity cannot however be supported in terms of hydrogen atom transfer taking into account
that the only hydrogen atom to consider for donation is that of the hydroxyl group, for which the abstraction is rather not easy (ΔBDE= + 4.0 kcal/mol). The high BDE value for sesamol in the gas-phase is in accordance with the theoretical findings of Najafi et al. [5]. Similar were the observations of Galano et al. [6] who calculated the free energy of activation for its reaction with various free radicals (●OH, ●OOH, ●OOCCl3) and found out that HAT was feasible only with the highly reactive ●OH in water. At this point it could be argued that either neutral sesamol acts preferably with a different mechanism or some structural modification is taking place during interaction with free radicals which enhances the antioxidant activity. The last group of compounds examined was a series of sesamin metabolites formed in the liver of mammals (Fig. 1B), some of which (M3, M6) are proposed to be very efficient and consequently responsible for the in vivo antioxidant potential of sesamin [3]. The first of the metabolites included in the study, M3, is this derived after opening of the 1,3-dibenzodioxole moiety resulting to the formation of a catechol moiety. Due to the presence of the latter structure the hydrogen atom donation was facilitated from the hydroxyl group at C-4’ (ΔBDE= -10.1 kcal/mol), considering also the phenoxy radical stabilization due to the formation of an intramolecular hydrogen bond with the adjacent hydroxyl group. Then a quinone was formed via the hydrogen atom abstraction at C-3’ (ΔBDE= -11.0 kcal/mol) and four hydrogen atoms from the fused five-member rings. The respective ΔBDE values for sequential four hydrogen atom able for donation were -7.7 (C-8), -31.6 (C-1), -23.0 (C-4) and -33.6 (C-5) kcal/mol respectively. Further structural changes resulting in the opening of the second 1,3-dibenzodioxole moiety, and consequently to the formation of an additional catechol moiety enhance the hydrogen atom donation efficiency. In such a case, the hydrogen atoms to be donated increase to eight, instead
of six found for M3. In M6 all the catechol hydrogen atoms are donated at first, thus, forming a di-quinone [ΔBDE= -10.0 (C-4”), -6.3 (C-4’), -14.3 (C-3”), -10.5 (C-3’) kcal/mol], followed by the other hydrogen atoms [ΔBDE= -20.4 (C-8), -19.8 (C-1), 18.6 (C-4), -37.2 (C-5) kcal/mol] in the same way as proposed for sesamin. After hydrogen atom donation M3 derivative was semi-planar, whereas the M6 one completely planar due to full conjugation. According to the ΔBDEsum values being equal to -117.0 and -137.1 kcal/mol for M3 and M6, respectively, it is obvious that these compounds are much more efficient hydrogen atom donors than sesamin, verifying, thus, the information reported by Wan et al. [3]. As polyhydroxy compounds are also toxic in vivo, structural modifications may progressively take place due to enzymatic activity. A common one is methoxylation resuling in M4 and M10. Thus, the antioxidant potential is expected to decrease since less hydrogen atoms are available for donation. Furthermore, after conversion to monophenolic compounds the non-phenolic hydrogen atoms become easier to donate, followed by only one phenolic from C-4’ [M4: ΔBDE= -7.81 (C-4), -31.4 (C-5), -8.0 (C-8), -44.9 (C-1), -10.3 (C-4’) kcal/mol, ΔBDEsum= -102.4 kcal/mol], [M10: ΔBDE= -5.4 (C-4), -0.4 (C-8), -45.1 (C-5), -43.5 (C-1), -7.2 (C-4’), +0.8 (C-4”) kcal/mol, ΔBDEsum= -101.6 kcal/mol]. The extension of conjugation can be achieved after hydrogen atom donation, even for M10 where except from the dimethylation, one of the furan rings is also open. Proceeding with metabolism, the opening of the second furan ring results in lower radical scavenging activity for M11 [ΔBDE= -0.7 (C-4’), -1.7 (C-4”) kcal/mol, ΔBDEsum= -2.4 kcal/mol). Enterodiol and enterolactone excreted in urine are rather inactive [M1: ΔBDE= +0.4 (C-3’) or +2.0 (C-3”), M2: ΔBDE= +2.3 (C-3’) or +2.2 (C-3”) kcal/mol] at least as hydrogen atom donors.
When calculations were carried out taking into consideration the solvent effect the computed ΔBDEsum values were not always affected significantly (Table 1) since in some cases the differences from those obtained in the gas phase were less than ± 1 kcal/mol. Nevertheless, for some compounds the differences were greater, especially with the computed values in water. More particularly, some of the compounds bearing phenolic group appeared to be more efficient in water. The latter could be related to improved stability conferred by the polar solvent to the parent and/or derived radicals as observed in the past for some flavonoids in methanol and water [9]. Even so, the hydrogen atom transfer trend of the examined compounds was the same regardless of the solvent effect consideration.
Table 1. Calculated ΔBDEsum values in benzene and water for the test compounds and difference from the gas-phase values (Δg-s) computed at 298 K and the B3LYP/6311++G (2d,2p)//B3LYP/6-311G level (kcal/mol) Benzene ΔBDEsum
water
Δg-s
ΔBDEsum
Δg-s
kcal/mol sesamin
-90.8
2.6
-90.3
3.1
sesamolin
-38.2
0.8
-38.4
0.6
episesamin
-89.7
2.3
-89.3
2.7
sesaminol
-102.7
-0.2
-105.7
-3.2
pinoresinol
-104.5
-0.2
-108.3
-4.0
sesamolinol
-46.4
0.7
-47.5
-0.4
sesamol
3.7
-0.3
2.4
-1.6
M1
0.9
-0.5
0.9
-0.5
M2
2
0.3
0.9
1.1
M3
-116.3
0.7
-119.7
-2.7
M4
-102.9
-0.5
-105.8
-4.4
M6
-137.4
-0.3
-143.2
-6.1
M10
-101.0
0.6
-104.9
-3.3
M11
-5.4
-3
-10.2
-7.8
The BDE values for phenol in benzene and water were 83.9 and 83.2 kcal/mol
3.3 Electron transfer
The values of the computed molecular descriptors related to electron transfer in the gas-phase and in solvent are presented in Table 2. Electron transfer to free radicals as predicted by the calculated IP values indicates a different trend in activity than that according to hydrogen atom transfer efficiency. This is not unusual since the IP values are affected by the whole structure of the test compound [30].
Table 2. Calculated values of molecular descriptors related to electron transfer at 298 K of test compounds at the B3LYP/6-311++G (2d,2p)//B3LYP/6-311G level in the gas-phase, benzene and water (kcal/mol). gas-phase IP
PA
ETE
benzene Δɷ±
IP
PA
ETE
water Δɷ±
IP
PA
ETE
Δɷ±
(kcal/mol) phenol
192.9
348.0
51.5
114.5
166.0
318.3
80.7
116.9
145.5
294.6
103.8
119.3
sesamin
162.3
-
-
109.1
145.9
-
-
111.5
133.4
-
-
114.1
sesamolin
160.4
-
-
110.0
143.8
-
-
111.7
129.4
-
-
113.6
episesamin
161.8
-
-
107.3
145.4
-
-
110.2
133.0
-
-
113.4
sesaminol
157.1
347.0
52.3
101.7
140.1
325.1
72.8
109.4
125.4
306.8
89.3
112.0
pinoresinol
161.5
345.8
52.7
103.1
145.6
318.9
77.3
104.9
133.5
296.2
97.6
107.9
sesamolinol
157.3
347.2
45.0
106.7
141.4
319.7
71.2
109.8
127.3
296.6
92.8
112.9
sesamol
171.3
347.2
56.4
102.7
146.5
318.7
84.1
106.6
127.7
295.6
105.9
109.6
M1
172.0
341.2
58.8
118.5
156.4
316.9
83.1
116.7
144.7
296.8
103.2
115.2
M2
179.8
339.8
62.0
126.0
159.0
316.2
84.9
126.3
142.6
295.6
103.0
128.7
M3
165.2
334.7
54.8
108.5
147.8
310.1
79.5
110.5
134.8
289.8
99.9
114.1
M4
162.0
348.9
50.5
107.7
146.0
321.0
76.0
110.7
133.8
297.1
97.4
113.7
M6
166.5
334.1
55.5
111.0
149.8
309.7
80.0
112.0
137.0
289.7
100.0
113.1
M10
160.6
347.2
51.7
102.8
145.0
320.0
75.9
103.4
133.1
298.2
95.7
105.6
M11
159.0
346.7
52.1
104.3
144.8
320.3
74.6
103.3
134.2
297.8
94.7
102.9
All the test compounds presented lower gas-phase IP values than that of phenol with ΔΙP ranging from -13.1 to – 35.8 kcal/mol. Sesaminol (ΔIP= -35.8 kcal/mol) and sesamolinol (ΔIP= -35.3 kcal/mol) were predicted to be the most efficient electron donors presenting values within the range of most flavonoids, that is 30-40 kcal/mol lower than that of phenol [32]. Conversion of sesamin to episesamin did not affect the electron donation in line with findings based on BDE values. Sesamol was found less efficient than the six examined lignans (ΔIP= -21.6 kcal/mol). Once again the higher radical scavenging activity proposed for such compound in comparison to that of sesamin or the comparable efficiency to pinoresinol [31] was not supported with computed gas-phase IP values. Poor electron donors, in case SET is favored, were also the M3 and M6 since their IP values differed from that of phenol less than 30 kcal/mol. Methylation of M3 and M6 improved the electron donating efficiency in line with past observations on catechol and guaiacol derivatives [9-11, 29]. The electron donating efficiency was adequate till the formation of M11 (ΔΙΡ= -33.9 kcal/mol). Nevertheless, when the M1 and M2 produced and excreted from the body a significant loss of electron donating efficiency was evidenced (ΔIP= -20.9 and -13.1 kcal/mol respectively). Another possible pathway to radical scavenging in polar media such as water or even alcohols is the SPLET mechanism [33]. A requirement is the deprotonation of the phenolic group which is characterized by the proton affinity (PA). The gas phase PA values for those of the studied compounds bearing a phenolic group are given in Table 2. From the respective compounds only M4 presents higher PA value from phenol by 0.9 kcal/mol. Among the lignans, pinoresinol is the one with the lowest PA value and, thus, able to deprotonate the phenolic group easier. Sesamol presents higher PA and ETE values in comparison to pinoresinol. Therefore, even SPLET mechanism cannot explain the experimental findings of Kuo et al. [31] in terms of gas-phase calculations. From the metabolites examined those that were more prone to deprotonation were the M3 and M6. In fact these compounds present the
lower gas-phase PA values from all tested molecules (ΔPA= -13.3 and -13.9 kcal/mol respectively). These values are comparable to that computed for hydroxytyrosol, a known bioactive phenol [13]. The corresponding ETE values were low, but not the lowest ones. However, if the sum of PA and ETE values is considered, the lowest one (390 kcal/mol) is calculated for M3 and M6. Thus, in polar media these compounds are expected to be the most efficient ones. Different efficiency can be expected only under pH conditions that will permit the deprotonation of compounds with higher PA values. The findings based on the calculation of the gas-phase net electrophilicity (Table 2) were not always in accordance with those based on gas-phase IP values. Thus, sesaminol which was predicted of equal electron donor efficiency to sesamolinol, according to IP values, seems better electron donor in terms of net electrophilicity. Furthermore, sesamol is found among the most electron donating compounds. Contradictory are the findings for sesamin and sesamolin also, whereas the results for M3 and M6 are consistent. Moreover, M1 and M2 presenting the higher IP values, were by far the most electron accepting of the test compounds on the basis of Δɷ± values. The metabolites M3 and M6 were the most prone to ionization and consequently more possible to act as efficient electron donors according to SPLET mechanism. When solvent was taken into account a drastic influence on IP values was observed because the charge separation process is sensitive to the solvent polarity [9]. Despite the fact that ionization and electron transfer is not expected in lipids, computed values were given just for comparison. Thus, differences were observed in terms of IP values and in certain cases the findings differed from those in the gas-phase. All compounds presented lower IP values from that of phenol, even if differences became progressively smaller by increasing solvent polarity. Still, sesaminol was predicted to be the most efficient donor and M1, M2 the least efficient ones. On the contrary, the neutral sesamol was found more efficient than
sesamin and pinoresinol. This can support the findings of Kuo et al. [31] for the reactivity of sesamol, granted that the experimental study was carried out in a polar environment which may support electron transfer. Regarding the SPLET pathway, clearly the ETE are lower than the corresponding IP values. However, the PA value is critical since it determines the ability of the molecule to form a phenolate ion. Thus, sesamol can be even more efficient electron donor in its ionic form, in agreement with the approach of Galano et al. [6] and their findings for neutral sesamol and its phenolate ion. However, since the pKa of the respective compound is 8.75 [6] it is difficult to act via SPLET under physiological conditions because the pH can be even lower than 7.4 in close proximity to the cell membranes [9], thus, not facilitating ionization. As shown, yet in water, the calculations support that M3 and M6 are more prone to the formation of phenolates, being therefore able to act even more efficiently as electron donors in case SPLET is favored over SET. Similarly, in the case of net electrophyllicity values, discrepancies and agreements were observed between the findings in water and gas-phase. M1 and M2 remained the most electron accepting compounds. Sesamol was an efficient electron donor but less than pinoresinol, a result contradicting the findings on the basis of gas-phase calculations.
4. Conclusions
The present study proposed for the first time that the major sesame oil lignans, namely sesamin and sesamolin may present antioxidant activity through hydrogen atom transfer, though lacking a phenolic group, as allylic hydrogen atoms seem able to be abstracted. In the case of tested compounds where a single phenolic group/s or guaiacol moiety/ies existed, allylic hydrogen atoms were usually more prone for abstraction than phenolic hydrogen atoms. This was not the case in metabolites bearing one or two catechol
moieties, where formation of quinone was preferable. Despite the contribution of nonphenolic hydrogen atoms to the antioxidant activity of lignans, still pinoresinol and metabolites having catechol moieties were more efficient than sesamin. The stepwise donation of non-phenolic hydrogen atoms affected conjugation and consequently the conformation of the compounds. Therefore, in many cases the proposed compounds to be formed are expected to be partially or completely planar, contrary to the mother compounds. BDE could not support for neutral sesamol a high antioxidant activity and particularly better or equal efficiency to that of some sesame oil lignans as found experimentally in the literature, even if the solvent effect was taken into account. Some of the tested compounds in terms of IP values may be of comparable potency to flavonoids with high electron donating efficiency. Neutral sesamol was found an efficient electron donor only when IP values were computed in water, justifying in part experimental findings. The latter finding was also supported by the net electrophilicity value calculated in water. Higher electron donating efficiency is rather not expected at a physiological pH due to phenolate formation since the pKa value is higher. The metabolites of sesamin M3 and M6, though week electron donors in their neutral form, are the most prone to ionization and consequently more capable to act then as efficient electron donors. This was verified even in water media. Thus, M3 and M6 can be potent antioxidants in both lipids (HAT dominance) or in polar media such as biological fluids (SPLET dominance). In this sense the enhancement of the in vivo antioxidant activity of sesamin can be justified. Finally, it has been shown that the excreted end products, enterolactone and enterodiol from the body of mammals are not expected to be efficient radical scavengers.
Supplementary Material
The supplementary material consists of a) the total energies, ZPE values and cartesian coordinates for the most stable conformers of the molecules under study (Table S1) b) the total energies and ZPE values for all the conformers of the molecules under study (Table S2). and c) the optimized structures of all the conformers in mol/mol2 .
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DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamine Anastasios G. Papadopoulos, Nikolaos Nenadis*, Michael P. Sigalas*
Captions to Figures
Fig. 1. Sesame lignans and sesamol (A) and sesamin metabolites (B) under study as cited in ref. [2]
Fig.2 Optimized structures of the molecules studied at the B3LYP/6-311G level (hydrogen atoms not shown for clarity)
Fig. 3. Proposed scavenging of free radicals by sesamin via sequential hydrogen atom donation
Fig. 4. Spin density distribution of sesaminol and pinoresinol phenoxy radicals
Figure 1A
Figure 1B
Figure 2
Figure 3
Figure 4
DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamine Anastasios G. Papadopoulos, Nikolaos Nenadis*, Michael P. Sigalas*
Table 1. Calculated ΔBDEsum values in benzene and water for the test compounds and difference from the gas-phase values (Δg-s) computed at 298 K and the B3LYP/6311++G (2d,2p)//B3LYP/6-311G level (kcal/mol) Benzene ΔBDEsum
water
Δg-s
ΔBDEsum
Δg-s
kcal/mol sesamin
-90.8
2.6
-90.3
3.1
sesamolin
-38.2
0.8
-38.4
0.6
episesamin
-89.7
2.3
-89.3
2.7
sesaminol
-102.7
-0.2
-105.7
-3.2
pinoresinol
-104.5
-0.2
-108.3
-4.0
sesamolinol
-46.4
0.7
-47.5
-0.4
sesamol
3.7
-0.3
2.4
-1.6
M1
0.9
-0.5
0.9
-0.5
M2
2
0.3
0.9
1.1
M3
-116.3
0.7
-119.7
-2.7
M4
-102.9
-0.5
-105.8
-4.4
M6
-137.4
-0.3
-143.2
-6.1
M10
-101.0
0.6
-104.9
-3.3
M11
-5.4
-3
-10.2
-7.8
The BDE values for phenol in benzene and water were 83.9 and 83.2 kcal/mol
Table 2. Calculated values of molecular descriptors related to electron transfer at 298 K of test compounds at the B3LYP/6-311++G (2d,2p)//B3LYP/6-311G level in the gas-phase, benzene and water (kcal/mol). gas-phase IP
PA
ETE
benzene Δɷ±
IP
PA
ETE
water Δɷ±
IP
PA
ETE
Δɷ±
(kcal/mol) phenol
192.9
348.0
51.5
114.5
166.0
318.3
80.7
116.9
145.5
294.6
103.8
119.3
sesamin
162.3
-
-
109.1
145.9
-
-
111.5
133.4
-
-
114.1
sesamolin
160.4
-
-
110.0
143.8
-
-
111.7
129.4
-
-
113.6
episesamin
161.8
-
-
107.3
145.4
-
-
110.2
133.0
-
-
113.4
sesaminol
157.1
347.0
52.3
101.7
140.1
325.1
72.8
109.4
125.4
306.8
89.3
112.0
pinoresinol
161.5
345.8
52.7
103.1
145.6
318.9
77.3
104.9
133.5
296.2
97.6
107.9
sesamolinol
157.3
347.2
45.0
106.7
141.4
319.7
71.2
109.8
127.3
296.6
92.8
112.9
sesamol
171.3
347.2
56.4
102.7
146.5
318.7
84.1
106.6
127.7
295.6
105.9
109.6
M1
172.0
341.2
58.8
118.5
156.4
316.9
83.1
116.7
144.7
296.8
103.2
115.2
M2
179.8
339.8
62.0
126.0
159.0
316.2
84.9
126.3
142.6
295.6
103.0
128.7
M3
165.2
334.7
54.8
108.5
147.8
310.1
79.5
110.5
134.8
289.8
99.9
114.1
M4
162.0
348.9
50.5
107.7
146.0
321.0
76.0
110.7
133.8
297.1
97.4
113.7
M6
166.5
334.1
55.5
111.0
149.8
309.7
80.0
112.0
137.0
289.7
100.0
113.1
M10
160.6
347.2
51.7
102.8
145.0
320.0
75.9
103.4
133.1
298.2
95.7
105.6
M11
159.0
346.7
52.1
104.3
144.8
320.3
74.6
103.3
134.2
297.8
94.7
102.9
Graphical Abstract
DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamine Anastasios G. Papadopoulos, Nikolaos Nenadis*, Michael P. Sigalas*
Highlights
The antioxidant activity of sesamine and related compounds were examined with DFT
Non phenolic hydrogen atoms can be transferred to free radicals
Reaction through hydrogen atom transfer affect molecular planarity
Sesamine in vivo metabolites with catechol group are the most efficient antioxidants
Sesamine metabolism lead to progressive loss of radical scavenging activity
http://dx.doi.org/10.1016/j.comptc.2015.11.016 COMPTC 1997
To appear in:
Computational & Theoretical Chemistry
Received Date: Revised Date: Accepted Date:
24 September 2015 16 November 2015 17 November 2015
Please cite this article as: A.G. Papadopoulos, N. Nenadis, M.P. Sigalas, DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamin, Computational & Theoretical Chemistry (2015), doi: http://dx.doi.org/10.1016/j.comptc.2015.11.016
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DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamin Anastasios G. Papadopoulosa, Nikolaos Nenadisb,*, Michael P. Sigalasa,**
a
Aristotle University of Thessaloniki, School of Chemistry, Laboratory of Applied
Quantum Chemistry, Thessaloniki 54124, Greece b
Aristotle University of Thessaloniki, School of Chemistry, Laboratory of Food
Chemistry and TEchnology, Thessaloniki 54124, Greece
**Corresponding author. Tel.: +30 2310 997815. E-mail address: [email protected] (M.P. Sigalas). * E-mail address: [email protected]
Abstract DFT calculation of various molecular descriptors was carried out in order to examine the radical scavenging properties of sesame oil lignans and some selected metabolites of sesamin formed in vivo. The major sesame oil lignans, namely sesamin and sesamolin may present antioxidant activity through hydrogen atom transfer, though lacking a phenolic group. This can be achieved via the contribution of allylic hydrogen atoms according to C-H bond dissociation enthalpy (BDE) values. Even so, the predicted activity is not higher than that of some phenolic derivatives (e.g. pinoresinol) or metabolites bearing catechol moieties. The contribution of nonphenolic hydrogen atoms may result in the formation of planar or semi-planar conjugated compounds. Some of the tested lignans (e.g. sesaminol, sesamolinol) may be efficient electron donors comparable to active flavonoids according to ionization potential (IP) values. Neutral sesamol can be an efficient electron donor rather than hydrogen atom donor according to IP and net electrophilicity values. Only computation in water could partially justify literature experimental findings on the higher reactivity of sesamol over that of sesamin. The metabolites of sesamin, bearing one or two catechol groups were found as the most efficient hydrogen atom donors and most prone to ionization. Therefore, they could be capable to act efficiently via hydrogen atom transfer (HAT) or sequential proton loss electron transfer (SPLET) in real systems as shown by gas-phase and calculations in solvents (benzene, water). Thus, these metabolites may account for the high in vivo antioxidant activity of sesamin. Examination of the succeeding products of metabolism indicated the progressive loss of radical scavenging efficiency which was predicted to be negligible in the compounds excreted from the body of mammals. Keywords: DFT; sesame oil; lignans; sesamin; pinoresinol; radical scavenging
1. Introduction Sesame seed is a pool of phytochemicals with significant nutritional importance and health-promoting properties [1]. Among these compounds, a set of furanolignans, namely sesamin, sesamolin and some minor ones (sesamol, sesaminol, sesamolinol, pinoresinol, matairesinol, lariciresinol and episesamin) have a broad spectrum of biological properties, including the suppression of lipid peroxidation [2,3]. The fat soluble ones, found in the sesame oil have been reported to contribute to the exceptional antioxidant activity of the oil, acting even synergistically with tocopherols and Maillard reaction products [3]. Due to their biological importance there is a raising interest in isolating lignans in high purity for medicinal applications [2] and even in the development of strategies aiming to obtain varieties with higher lignan content [1]. In this way a crop of higher nutritional and functional value may be obtained, which can produce edible oil with higher shelf-life and added value. Despite the fact that sesame lignans are of high importance for the food and pharmaceutical sector, there is limited theoretical information concerning their electronic properties, and particularly those related to their radical scavenging activity. The latter is possibly due to the fact that major sesame lignans do not bear any hydroxyl group, whereas on the basis of some in vitro experiments are considered rather poor antioxidants, although there are contradictory findings [3]. Thus, information available regards pinoresinol [4], a derivative of sesamin hydrolysis during oil production, or sesamol [5, 6] deriving from sesamolin under the same conditions, both of which bear a hydroxyl group attached to the aromatic ring. Other theoretical studies, on e.g. pinoresinol are related to conformation analysis [7]. Taking into account the above, B3LYP density functional theory calculations were used to investigate the structure-radical scavenging activity of major and minor
sesame lignans (Fig. 1A) Furthermore, the study has been extended to a set of metabolites of sesamin which can be formed in the liver of mammals, together with those forms eventually excreted in their urine (Fig. 1B). Some of these metabolites due to the presence of catechol moieties have been considered as the active forms responsible for the in vivo antioxidant effect of lignans [3].
Fig. 1 Sesame lignans and sesamol (A), and its in vivo metabolites (B) under study as cited in ref [2].
A series of molecular descriptors (bond dissociation enthalpy, BDE; ionization potential, IP; proton dissociation enthalpy, PDE; proton affinity, PA; electron transfer energy, ETE; net electrophilicity, Δɷ±) characterizing the hydrogen atom or electron donating efficiency of the selected compounds were computed [8-11]. Calculations were first carried out in the gas-phase as good primary indices for radical scavenging [12, 13]. Solute–solvent interactions were also considered using theoretical models
approximating the activity of test compounds in lipid or in aqueous media. With the current study we expect to add to the characterization of the antioxidant properties of the respective compounds and stimulate the interest for further studies and exploitation by the food and pharmaceutical industries.
2. Computational details
All calculations were performed by the Gaussian 03W ver. 6.0 set of programs [14]. The hybrid DFT method with Becke’s three-parameter functional [15] and the nonlocal correlation provided by the Lee, Yang, Parr expression [16] (B3LYP) was used for geometry optimization using the 6-311G basis set (unrestricted B3LYP for the optimization of the resulting radicals). Frequency calculations were performed at the same level of theory to characterize the nature of the stationary points. Singlepoint electronic energies were then obtained using the 6-311++G (2d,2p) basis set. Employing the electronic energies (298 K) at 6-311++G (2d,2p) and thermal contributions to enthalpy obtained at 6-311G, the bond dissociation enthalpy (BDE) values that characterize hydrogen atom donating activity (HAT) were determined according to the equation: BDE = Hr + Hh – Hp, where Hr is the enthalpy of the radical generated by H-atom abstraction, Hh is the enthalpy of the H-atom (−0.499897 hartree at this level of theory), and Hp is the enthalpy of the parent molecule. In the examination of sequential loss of hydrogen atoms, BDE was calculated taking into account the appropriate species. The ionization potential (IP) values characterizing single electron (SET) transfer efficiency were determined according to the equation: IP = Ecr − Ep, where p and cr stand for the parent molecule and the corresponding cation radical generated after electron transfer. Proton affinity
(PA) values defining the first step of sequential proton loss electron transfer (SPLET) mechanism were calculated according to the formula: PA = Ha + Hpr − Hp, where Ha is the enthalpy of the anion generated after deprotonation, Hpr is the enthalpy of the proton (0.00236 hartree), and Hp is the enthalpy of the parent molecule. The electron transfer energy (ETE) or otherwise ionization potential values of the anions were obtained via the equation: ETE = Er – Ea, where r and a are referred to the phenoxy radical and the anion, respectively. No spin contamination was found for radicals, with the
3. Results and discussion 3.1. Conformation analysis To compute the appropriate values of the selected molecular descriptors that may characterize the antioxidant activity of the compounds, the conformational space of the molecules under investigation has been explored in order to locate the structure of the global minimum among all the possible conformers. For each molecule a series of
conformations being minima with energies close to this of the global minimum of the potential energy surface have been located. Thus, 2 local minima in an energy window of 11.4 kcal/mol above the global minimum have been found for sesamin, 1 in 1.5 kcal/mol for epipesamin, 7 in 5.3 kcal/mol for sesamolin, 12 in 14.5 kcal/mol for sesaminol, 4 in 2.9 kcal/mol for sesamolinol, 5 in 13.7 kcal/mol for pinoresinol, 1 in 0.3 kcal/mol for sesamol, 9 in 7.5 kcal/mol for M3, 5 in 7.9 kcal/mol for M4, 11 in 12.8 kcal/mol for M6, 2 in 10.6 kcal/mol for M10, 1 in 5.3 kcal/mol for M11, 2 in 0.3 kcal/mol for M1 and 1 in 0.2 kcal/mol for M2. The optimized structure of the global minimum for all the lignans, sesamol and sesamin metabolites are presented in Fig. 2. In all molecules bearing dioxabicyclooctane system, the two five-membered rings are cis-fused and furan ring adopts a half-chair conformation. There is a satisfactory agreement between the calculated structural parameters and those derived from X-ray studies for sesamin [18-20], episesamin [20-23], sesaminol [24], pinoresinol [25] and lariciresinol [25]. The local minima found have the same conformation of the dioxabicyclooctane system and differ in the tortion angle defining the orientation of the benzodioxole ring.
Fig.2. Optimized structures of the molecules studied at the B3LYP/6-311G level (hydrogen atoms not shown for clarity and are given as supplementary material) 3.2. Hydrogen atom transfer In the process of radical scavenging activity, the number and the relative position of hydroxyl groups in phenolic compounds are important [26]. However, on the basis of quantum chemical calculations it has been suggested that allylic hydrogen atoms may also participate to the scavenging of free radicals. In some cases, namely in catechins, such hydrogen atoms may compete with those of the hydroxyl groups [27], whereas in other cases they may follow in donation as it has been predicted for dihydroxy-caffeic acid [26] or some synthetic isochroman derivatives of hydroxytyrosol [13]. A non phenolic hydrogen atom, other than allylic, has recently been proposed to be of significant importance for the radical scavenging of oleuropein according to DFT calculations [28]. Due to the lack of hydroxyl group in the major sesame lignans, namely sesamin and sesamolin, inevitably calculations were focused on the possible donation of allylic hydrogen atoms. Thus, it was found that in sesamin the allylic hydrogen atom at C-4 can be abstracted considering the difference in BDE value (ΔBDE= -7.9 kcal/mol) calculated relatively to that of the hydroxyl group of phenol (BDE= 84.4 kcal/mol). Phenol is expected to be inactive, unless the free radical is very drastic (e.g. ●
OH), and it may serve as a reference compound [9]. Furthermore, the BDE value for
the allylic hydrogen is only 1.5 kcal/mol higher than that computed for catechol (75.2 kcal/mol), an essential structural feature for adequate antioxidant activity via HAT. Trying to examine further the antioxidant potential of sesamin when the HAT is dominant, we have explored a possible mechanism depicted in Fig. 3. For each step
the energetically favored abstraction was located by optimizing radicals derived by all the possible hydrogen donated.
Fig. 3. Proposed scavenging of free radicals by sesamin via sequential hydrogen atom donation
It seems that after the allylic atom abstraction, the donation of a hydrogen atom from a vicinal carbon at C-5 is feasible so that the molecule to become stable via the formation of a double bond. The corresponding value is too low in comparison to that of phenol (ΔBDE= -34.0 kcal/mol), which is rather expected considering that the derived molecule is more stable. The value is of the same magnitude to that calculated for other C-H bonds with low BDE which can participate in reaction eventually leading to more stable structure [10, 29]. Considering the symmetry of the compound a second allylic hydrogen atom seems possible for abstraction at C-8 (ΔBDE= -6.3 kcal/mol), followed by a second hydrogen atom abstraction from the neighboring carbon C-1 (ΔBDE= -45.0 kcal/mol), resulting in a second double bond formation. The BDE value at C-8’ was found considerably higher than that of phenol (ΔBDE= +5.1 kcal/mol). Thus, it is not expected to add to the antioxidant potency of the compound, although studies calculating energies of activation or rate constants with individual radical can be useful to clarify this [6]. Consequently sesamin seems to be able to donate four hydrogen atoms with a sum of the four ΔΒDE values equal to -
93.4 kcal/mol. The ΔΒDEsum was used as a measure of the total relative to phenol enthalpy resulting from the contribution of all hydrogen atoms that could be donated by the test compound. The proposed mechanism affects the molecular geometry, as the formation of double bonds results in a conjugated planar molecule, contrary to the mother compound (sesamin). Similar findings to those evidenced for sesamin were observed for sesamolin regarding the part of the molecule that bears an allylic hydrogen. Thus, ΔBDE values calculated for the abstraction of the corresponding hydrogen at C-8 and the subsequent formation of a double bond after abstraction at C-1 were -7.8 and -31.3 kcal/mol respectively. Nevertheless, in the other part of the molecule, the introduction of an oxygen group between the furan and the aromatic ring changes the electronic phenomena. Therefore, the C-H BDE value at C-4 became higher than that computed for phenol (ΔBDE +7.1 kcal/mol). Consequently, sesamolin is expected to be less efficient than sesamin, considering that it may donate only two hydrogen atoms (ΔBDEsum= -39.0 kcal/mol). As already mentioned for sesamin, the formation of a double bond is expected to affect the geometry of the molecule making it partially planar. Conversion of sesamin to its geometrical isomer (episesamin) during refining of the oil is not expected to affect significantly the BDE values. This is likely taking into account that the calculated values are affected by the local phenomena rather than the whole structure [30]. Indeed the ΔBDEsum= -92.0 kcal/mol, that is only 1.4 kcal/mol higher than that of sesamin. Steric effects on the other hand may influence the interaction with the free radicals and therefore the radical scavenging efficiency. Molecules that derive from the major lignans during the oil production and bear a phenolic group, namely sesaminol, pinoresinol and sesamolinol were then
examined. In the case of sesaminol, the abstraction of the allylic hydrogen atom at C-6' followed by formation of a double bond was preferable than that of the phenolic hydrogen atom. The obtained values were almost comparable to those of sesamin (ΔBDEsum= -93. 0 kcal/mol). In addition, the hydrogen atom donation from the phenolic group which was not favored as a primary site of donation (84.4 kcal/mol), was now predicted to be easily prone to abstraction (ΔBDE= -9.4 kcal/mol). The latter is probably due to the extended conjugation formed in the compound which stabilizes more effectively the derived phenoxy radical. This is illustrated clearly in Fig. 4, where spin delocalization takes place not only in the aromatic ring, as it would be in case the phenolic hydrogen atom is abstracted first, but also through both the fused five- member rings. As a consequence the BDE value significantly becomes lower. Thus, sesaminol is expected to be more efficient than sesamin, considering that the ΔBDEsum for the five hydrogen atoms was -102.5 kcal/mol. In pinoresinol, the same tendency was observed, leading to a planar and fully conjugated molecule. After its formation, a phenolic hydrogen atoms could further be abstracted easily at C-4' (ΔBDE= -10.5 kcal/mol). This is probably due to the extended conjugation of the compound (Fig. 4), as previously proposed for sesaminol.
Fig. 4. Spin density distribution of sesaminol and pinoresinol phenoxy radicals
However, donation of the second one may be rather not feasible (ΔBDE= +0.9 kcal/mol), at least with not very reactive free radicals, despite the fact that the quinone (paired-electron system) should be more stable than a phenoxy radical (unpaired electron system). The respective approach indicated that pinoresinol should be much more efficient radical scavenger than predicted in the past where calculations only considered the BDE value of the phenolic groups [4]. Furthermore, it should be more efficient than sesamin (ΔBDEsum= -104.3 kcal/mol) in accordance with experimental findings [31]. Contrary to what observed in the above two compounds, the primary hydrogen atom for donation in sesamolinol was found to be this of the phenolic group at C-4" (ΔBDE= -7.4 kcal/mol). This is rather surprising considering that sequential hydrogen atom donation starting from the allylic hydrogen atom donation from C-4 was still feasible (ΔBDE= -5.2 kcal/mol), despite the fact that an unstable system bearing two un-paired electrons would be formed in this way. Even so, a rapid hydrogen atom donation from the vicinal carbon at C-5 could follow (ΔBDE= -34.4 kcal/mol) leading to a more stable phenoxy radical. Similarly to sesamolin, the hydrogen atom at C-8 seems not feasible for donation (ΔBDE= +8.3 kcal/mol). Nevertheless, sesamolinol is expected to be more potent than sesamolin due to the presence of the phenol group (ΔBDE= -47.1 kcal/mol). Sesamol is the simplest compound deriving from sesamolin hydrolysis during roasting. It is considered essentially an efficient radical scavenger using various in vitro assays as summarized by Wan et al. [3]. As a matter of fact it has been found more efficient in the scavenging of DPPH● and superoxide anion than sesamin and sesamolin, and of comparable activity to that of pinoresinol [31]. Such an activity cannot however be supported in terms of hydrogen atom transfer taking into account
that the only hydrogen atom to consider for donation is that of the hydroxyl group, for which the abstraction is rather not easy (ΔBDE= + 4.0 kcal/mol). The high BDE value for sesamol in the gas-phase is in accordance with the theoretical findings of Najafi et al. [5]. Similar were the observations of Galano et al. [6] who calculated the free energy of activation for its reaction with various free radicals (●OH, ●OOH, ●OOCCl3) and found out that HAT was feasible only with the highly reactive ●OH in water. At this point it could be argued that either neutral sesamol acts preferably with a different mechanism or some structural modification is taking place during interaction with free radicals which enhances the antioxidant activity. The last group of compounds examined was a series of sesamin metabolites formed in the liver of mammals (Fig. 1B), some of which (M3, M6) are proposed to be very efficient and consequently responsible for the in vivo antioxidant potential of sesamin [3]. The first of the metabolites included in the study, M3, is this derived after opening of the 1,3-dibenzodioxole moiety resulting to the formation of a catechol moiety. Due to the presence of the latter structure the hydrogen atom donation was facilitated from the hydroxyl group at C-4’ (ΔBDE= -10.1 kcal/mol), considering also the phenoxy radical stabilization due to the formation of an intramolecular hydrogen bond with the adjacent hydroxyl group. Then a quinone was formed via the hydrogen atom abstraction at C-3’ (ΔBDE= -11.0 kcal/mol) and four hydrogen atoms from the fused five-member rings. The respective ΔBDE values for sequential four hydrogen atom able for donation were -7.7 (C-8), -31.6 (C-1), -23.0 (C-4) and -33.6 (C-5) kcal/mol respectively. Further structural changes resulting in the opening of the second 1,3-dibenzodioxole moiety, and consequently to the formation of an additional catechol moiety enhance the hydrogen atom donation efficiency. In such a case, the hydrogen atoms to be donated increase to eight, instead
of six found for M3. In M6 all the catechol hydrogen atoms are donated at first, thus, forming a di-quinone [ΔBDE= -10.0 (C-4”), -6.3 (C-4’), -14.3 (C-3”), -10.5 (C-3’) kcal/mol], followed by the other hydrogen atoms [ΔBDE= -20.4 (C-8), -19.8 (C-1), 18.6 (C-4), -37.2 (C-5) kcal/mol] in the same way as proposed for sesamin. After hydrogen atom donation M3 derivative was semi-planar, whereas the M6 one completely planar due to full conjugation. According to the ΔBDEsum values being equal to -117.0 and -137.1 kcal/mol for M3 and M6, respectively, it is obvious that these compounds are much more efficient hydrogen atom donors than sesamin, verifying, thus, the information reported by Wan et al. [3]. As polyhydroxy compounds are also toxic in vivo, structural modifications may progressively take place due to enzymatic activity. A common one is methoxylation resuling in M4 and M10. Thus, the antioxidant potential is expected to decrease since less hydrogen atoms are available for donation. Furthermore, after conversion to monophenolic compounds the non-phenolic hydrogen atoms become easier to donate, followed by only one phenolic from C-4’ [M4: ΔBDE= -7.81 (C-4), -31.4 (C-5), -8.0 (C-8), -44.9 (C-1), -10.3 (C-4’) kcal/mol, ΔBDEsum= -102.4 kcal/mol], [M10: ΔBDE= -5.4 (C-4), -0.4 (C-8), -45.1 (C-5), -43.5 (C-1), -7.2 (C-4’), +0.8 (C-4”) kcal/mol, ΔBDEsum= -101.6 kcal/mol]. The extension of conjugation can be achieved after hydrogen atom donation, even for M10 where except from the dimethylation, one of the furan rings is also open. Proceeding with metabolism, the opening of the second furan ring results in lower radical scavenging activity for M11 [ΔBDE= -0.7 (C-4’), -1.7 (C-4”) kcal/mol, ΔBDEsum= -2.4 kcal/mol). Enterodiol and enterolactone excreted in urine are rather inactive [M1: ΔBDE= +0.4 (C-3’) or +2.0 (C-3”), M2: ΔBDE= +2.3 (C-3’) or +2.2 (C-3”) kcal/mol] at least as hydrogen atom donors.
When calculations were carried out taking into consideration the solvent effect the computed ΔBDEsum values were not always affected significantly (Table 1) since in some cases the differences from those obtained in the gas phase were less than ± 1 kcal/mol. Nevertheless, for some compounds the differences were greater, especially with the computed values in water. More particularly, some of the compounds bearing phenolic group appeared to be more efficient in water. The latter could be related to improved stability conferred by the polar solvent to the parent and/or derived radicals as observed in the past for some flavonoids in methanol and water [9]. Even so, the hydrogen atom transfer trend of the examined compounds was the same regardless of the solvent effect consideration.
Table 1. Calculated ΔBDEsum values in benzene and water for the test compounds and difference from the gas-phase values (Δg-s) computed at 298 K and the B3LYP/6311++G (2d,2p)//B3LYP/6-311G level (kcal/mol) Benzene ΔBDEsum
water
Δg-s
ΔBDEsum
Δg-s
kcal/mol sesamin
-90.8
2.6
-90.3
3.1
sesamolin
-38.2
0.8
-38.4
0.6
episesamin
-89.7
2.3
-89.3
2.7
sesaminol
-102.7
-0.2
-105.7
-3.2
pinoresinol
-104.5
-0.2
-108.3
-4.0
sesamolinol
-46.4
0.7
-47.5
-0.4
sesamol
3.7
-0.3
2.4
-1.6
M1
0.9
-0.5
0.9
-0.5
M2
2
0.3
0.9
1.1
M3
-116.3
0.7
-119.7
-2.7
M4
-102.9
-0.5
-105.8
-4.4
M6
-137.4
-0.3
-143.2
-6.1
M10
-101.0
0.6
-104.9
-3.3
M11
-5.4
-3
-10.2
-7.8
The BDE values for phenol in benzene and water were 83.9 and 83.2 kcal/mol
3.3 Electron transfer
The values of the computed molecular descriptors related to electron transfer in the gas-phase and in solvent are presented in Table 2. Electron transfer to free radicals as predicted by the calculated IP values indicates a different trend in activity than that according to hydrogen atom transfer efficiency. This is not unusual since the IP values are affected by the whole structure of the test compound [30].
Table 2. Calculated values of molecular descriptors related to electron transfer at 298 K of test compounds at the B3LYP/6-311++G (2d,2p)//B3LYP/6-311G level in the gas-phase, benzene and water (kcal/mol). gas-phase IP
PA
ETE
benzene Δɷ±
IP
PA
ETE
water Δɷ±
IP
PA
ETE
Δɷ±
(kcal/mol) phenol
192.9
348.0
51.5
114.5
166.0
318.3
80.7
116.9
145.5
294.6
103.8
119.3
sesamin
162.3
-
-
109.1
145.9
-
-
111.5
133.4
-
-
114.1
sesamolin
160.4
-
-
110.0
143.8
-
-
111.7
129.4
-
-
113.6
episesamin
161.8
-
-
107.3
145.4
-
-
110.2
133.0
-
-
113.4
sesaminol
157.1
347.0
52.3
101.7
140.1
325.1
72.8
109.4
125.4
306.8
89.3
112.0
pinoresinol
161.5
345.8
52.7
103.1
145.6
318.9
77.3
104.9
133.5
296.2
97.6
107.9
sesamolinol
157.3
347.2
45.0
106.7
141.4
319.7
71.2
109.8
127.3
296.6
92.8
112.9
sesamol
171.3
347.2
56.4
102.7
146.5
318.7
84.1
106.6
127.7
295.6
105.9
109.6
M1
172.0
341.2
58.8
118.5
156.4
316.9
83.1
116.7
144.7
296.8
103.2
115.2
M2
179.8
339.8
62.0
126.0
159.0
316.2
84.9
126.3
142.6
295.6
103.0
128.7
M3
165.2
334.7
54.8
108.5
147.8
310.1
79.5
110.5
134.8
289.8
99.9
114.1
M4
162.0
348.9
50.5
107.7
146.0
321.0
76.0
110.7
133.8
297.1
97.4
113.7
M6
166.5
334.1
55.5
111.0
149.8
309.7
80.0
112.0
137.0
289.7
100.0
113.1
M10
160.6
347.2
51.7
102.8
145.0
320.0
75.9
103.4
133.1
298.2
95.7
105.6
M11
159.0
346.7
52.1
104.3
144.8
320.3
74.6
103.3
134.2
297.8
94.7
102.9
All the test compounds presented lower gas-phase IP values than that of phenol with ΔΙP ranging from -13.1 to – 35.8 kcal/mol. Sesaminol (ΔIP= -35.8 kcal/mol) and sesamolinol (ΔIP= -35.3 kcal/mol) were predicted to be the most efficient electron donors presenting values within the range of most flavonoids, that is 30-40 kcal/mol lower than that of phenol [32]. Conversion of sesamin to episesamin did not affect the electron donation in line with findings based on BDE values. Sesamol was found less efficient than the six examined lignans (ΔIP= -21.6 kcal/mol). Once again the higher radical scavenging activity proposed for such compound in comparison to that of sesamin or the comparable efficiency to pinoresinol [31] was not supported with computed gas-phase IP values. Poor electron donors, in case SET is favored, were also the M3 and M6 since their IP values differed from that of phenol less than 30 kcal/mol. Methylation of M3 and M6 improved the electron donating efficiency in line with past observations on catechol and guaiacol derivatives [9-11, 29]. The electron donating efficiency was adequate till the formation of M11 (ΔΙΡ= -33.9 kcal/mol). Nevertheless, when the M1 and M2 produced and excreted from the body a significant loss of electron donating efficiency was evidenced (ΔIP= -20.9 and -13.1 kcal/mol respectively). Another possible pathway to radical scavenging in polar media such as water or even alcohols is the SPLET mechanism [33]. A requirement is the deprotonation of the phenolic group which is characterized by the proton affinity (PA). The gas phase PA values for those of the studied compounds bearing a phenolic group are given in Table 2. From the respective compounds only M4 presents higher PA value from phenol by 0.9 kcal/mol. Among the lignans, pinoresinol is the one with the lowest PA value and, thus, able to deprotonate the phenolic group easier. Sesamol presents higher PA and ETE values in comparison to pinoresinol. Therefore, even SPLET mechanism cannot explain the experimental findings of Kuo et al. [31] in terms of gas-phase calculations. From the metabolites examined those that were more prone to deprotonation were the M3 and M6. In fact these compounds present the
lower gas-phase PA values from all tested molecules (ΔPA= -13.3 and -13.9 kcal/mol respectively). These values are comparable to that computed for hydroxytyrosol, a known bioactive phenol [13]. The corresponding ETE values were low, but not the lowest ones. However, if the sum of PA and ETE values is considered, the lowest one (390 kcal/mol) is calculated for M3 and M6. Thus, in polar media these compounds are expected to be the most efficient ones. Different efficiency can be expected only under pH conditions that will permit the deprotonation of compounds with higher PA values. The findings based on the calculation of the gas-phase net electrophilicity (Table 2) were not always in accordance with those based on gas-phase IP values. Thus, sesaminol which was predicted of equal electron donor efficiency to sesamolinol, according to IP values, seems better electron donor in terms of net electrophilicity. Furthermore, sesamol is found among the most electron donating compounds. Contradictory are the findings for sesamin and sesamolin also, whereas the results for M3 and M6 are consistent. Moreover, M1 and M2 presenting the higher IP values, were by far the most electron accepting of the test compounds on the basis of Δɷ± values. The metabolites M3 and M6 were the most prone to ionization and consequently more possible to act as efficient electron donors according to SPLET mechanism. When solvent was taken into account a drastic influence on IP values was observed because the charge separation process is sensitive to the solvent polarity [9]. Despite the fact that ionization and electron transfer is not expected in lipids, computed values were given just for comparison. Thus, differences were observed in terms of IP values and in certain cases the findings differed from those in the gas-phase. All compounds presented lower IP values from that of phenol, even if differences became progressively smaller by increasing solvent polarity. Still, sesaminol was predicted to be the most efficient donor and M1, M2 the least efficient ones. On the contrary, the neutral sesamol was found more efficient than
sesamin and pinoresinol. This can support the findings of Kuo et al. [31] for the reactivity of sesamol, granted that the experimental study was carried out in a polar environment which may support electron transfer. Regarding the SPLET pathway, clearly the ETE are lower than the corresponding IP values. However, the PA value is critical since it determines the ability of the molecule to form a phenolate ion. Thus, sesamol can be even more efficient electron donor in its ionic form, in agreement with the approach of Galano et al. [6] and their findings for neutral sesamol and its phenolate ion. However, since the pKa of the respective compound is 8.75 [6] it is difficult to act via SPLET under physiological conditions because the pH can be even lower than 7.4 in close proximity to the cell membranes [9], thus, not facilitating ionization. As shown, yet in water, the calculations support that M3 and M6 are more prone to the formation of phenolates, being therefore able to act even more efficiently as electron donors in case SPLET is favored over SET. Similarly, in the case of net electrophyllicity values, discrepancies and agreements were observed between the findings in water and gas-phase. M1 and M2 remained the most electron accepting compounds. Sesamol was an efficient electron donor but less than pinoresinol, a result contradicting the findings on the basis of gas-phase calculations.
4. Conclusions
The present study proposed for the first time that the major sesame oil lignans, namely sesamin and sesamolin may present antioxidant activity through hydrogen atom transfer, though lacking a phenolic group, as allylic hydrogen atoms seem able to be abstracted. In the case of tested compounds where a single phenolic group/s or guaiacol moiety/ies existed, allylic hydrogen atoms were usually more prone for abstraction than phenolic hydrogen atoms. This was not the case in metabolites bearing one or two catechol
moieties, where formation of quinone was preferable. Despite the contribution of nonphenolic hydrogen atoms to the antioxidant activity of lignans, still pinoresinol and metabolites having catechol moieties were more efficient than sesamin. The stepwise donation of non-phenolic hydrogen atoms affected conjugation and consequently the conformation of the compounds. Therefore, in many cases the proposed compounds to be formed are expected to be partially or completely planar, contrary to the mother compounds. BDE could not support for neutral sesamol a high antioxidant activity and particularly better or equal efficiency to that of some sesame oil lignans as found experimentally in the literature, even if the solvent effect was taken into account. Some of the tested compounds in terms of IP values may be of comparable potency to flavonoids with high electron donating efficiency. Neutral sesamol was found an efficient electron donor only when IP values were computed in water, justifying in part experimental findings. The latter finding was also supported by the net electrophilicity value calculated in water. Higher electron donating efficiency is rather not expected at a physiological pH due to phenolate formation since the pKa value is higher. The metabolites of sesamin M3 and M6, though week electron donors in their neutral form, are the most prone to ionization and consequently more capable to act then as efficient electron donors. This was verified even in water media. Thus, M3 and M6 can be potent antioxidants in both lipids (HAT dominance) or in polar media such as biological fluids (SPLET dominance). In this sense the enhancement of the in vivo antioxidant activity of sesamin can be justified. Finally, it has been shown that the excreted end products, enterolactone and enterodiol from the body of mammals are not expected to be efficient radical scavengers.
Supplementary Material
The supplementary material consists of a) the total energies, ZPE values and cartesian coordinates for the most stable conformers of the molecules under study (Table S1) b) the total energies and ZPE values for all the conformers of the molecules under study (Table S2). and c) the optimized structures of all the conformers in mol/mol2 .
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DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamine Anastasios G. Papadopoulos, Nikolaos Nenadis*, Michael P. Sigalas*
Captions to Figures
Fig. 1. Sesame lignans and sesamol (A) and sesamin metabolites (B) under study as cited in ref. [2]
Fig.2 Optimized structures of the molecules studied at the B3LYP/6-311G level (hydrogen atoms not shown for clarity)
Fig. 3. Proposed scavenging of free radicals by sesamin via sequential hydrogen atom donation
Fig. 4. Spin density distribution of sesaminol and pinoresinol phenoxy radicals
Figure 1A
Figure 1B
Figure 2
Figure 3
Figure 4
DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamine Anastasios G. Papadopoulos, Nikolaos Nenadis*, Michael P. Sigalas*
Table 1. Calculated ΔBDEsum values in benzene and water for the test compounds and difference from the gas-phase values (Δg-s) computed at 298 K and the B3LYP/6311++G (2d,2p)//B3LYP/6-311G level (kcal/mol) Benzene ΔBDEsum
water
Δg-s
ΔBDEsum
Δg-s
kcal/mol sesamin
-90.8
2.6
-90.3
3.1
sesamolin
-38.2
0.8
-38.4
0.6
episesamin
-89.7
2.3
-89.3
2.7
sesaminol
-102.7
-0.2
-105.7
-3.2
pinoresinol
-104.5
-0.2
-108.3
-4.0
sesamolinol
-46.4
0.7
-47.5
-0.4
sesamol
3.7
-0.3
2.4
-1.6
M1
0.9
-0.5
0.9
-0.5
M2
2
0.3
0.9
1.1
M3
-116.3
0.7
-119.7
-2.7
M4
-102.9
-0.5
-105.8
-4.4
M6
-137.4
-0.3
-143.2
-6.1
M10
-101.0
0.6
-104.9
-3.3
M11
-5.4
-3
-10.2
-7.8
The BDE values for phenol in benzene and water were 83.9 and 83.2 kcal/mol
Table 2. Calculated values of molecular descriptors related to electron transfer at 298 K of test compounds at the B3LYP/6-311++G (2d,2p)//B3LYP/6-311G level in the gas-phase, benzene and water (kcal/mol). gas-phase IP
PA
ETE
benzene Δɷ±
IP
PA
ETE
water Δɷ±
IP
PA
ETE
Δɷ±
(kcal/mol) phenol
192.9
348.0
51.5
114.5
166.0
318.3
80.7
116.9
145.5
294.6
103.8
119.3
sesamin
162.3
-
-
109.1
145.9
-
-
111.5
133.4
-
-
114.1
sesamolin
160.4
-
-
110.0
143.8
-
-
111.7
129.4
-
-
113.6
episesamin
161.8
-
-
107.3
145.4
-
-
110.2
133.0
-
-
113.4
sesaminol
157.1
347.0
52.3
101.7
140.1
325.1
72.8
109.4
125.4
306.8
89.3
112.0
pinoresinol
161.5
345.8
52.7
103.1
145.6
318.9
77.3
104.9
133.5
296.2
97.6
107.9
sesamolinol
157.3
347.2
45.0
106.7
141.4
319.7
71.2
109.8
127.3
296.6
92.8
112.9
sesamol
171.3
347.2
56.4
102.7
146.5
318.7
84.1
106.6
127.7
295.6
105.9
109.6
M1
172.0
341.2
58.8
118.5
156.4
316.9
83.1
116.7
144.7
296.8
103.2
115.2
M2
179.8
339.8
62.0
126.0
159.0
316.2
84.9
126.3
142.6
295.6
103.0
128.7
M3
165.2
334.7
54.8
108.5
147.8
310.1
79.5
110.5
134.8
289.8
99.9
114.1
M4
162.0
348.9
50.5
107.7
146.0
321.0
76.0
110.7
133.8
297.1
97.4
113.7
M6
166.5
334.1
55.5
111.0
149.8
309.7
80.0
112.0
137.0
289.7
100.0
113.1
M10
160.6
347.2
51.7
102.8
145.0
320.0
75.9
103.4
133.1
298.2
95.7
105.6
M11
159.0
346.7
52.1
104.3
144.8
320.3
74.6
103.3
134.2
297.8
94.7
102.9
Graphical Abstract
DFT study of radical scavenging activity of sesame oil lignans and selected in vivo metabolites of sesamine Anastasios G. Papadopoulos, Nikolaos Nenadis*, Michael P. Sigalas*
Highlights
The antioxidant activity of sesamine and related compounds were examined with DFT
Non phenolic hydrogen atoms can be transferred to free radicals
Reaction through hydrogen atom transfer affect molecular planarity
Sesamine in vivo metabolites with catechol group are the most efficient antioxidants
Sesamine metabolism lead to progressive loss of radical scavenging activity