The hydroperoxyl radical scavenging activity of sulfuretin: insights from theory.

Sulfuretin (SFR), which is isolated from Rhus verniciflua, Toxicodendron vernicifluum, Dahlia, Bidens tripartite and Dipterx lacunifera, is one of the most important natural flavonoids. This compound is known to have numerous biological activities; among these, the antioxidant activity has not been thoroughly studied yet. In this study, the hydroperoxyl scavenging activity of SFR was examined by using density functional theory calculations. SFR is predicted to be an excellent HOO• scavenger in water at pH = 7.40 with k overall = 4.75 × 107 M-1 s-1, principally due to an increase in the activity of the anionic form following the single-electron transfer mechanism. Consistently, the activity of the neutral form is more prominent in the non-polar environment with k overall = 1.79 × 104 M-1 s-1 following the formal hydrogen transfer mechanism. Thus, it is predicted that SFR exhibits better HOO• antiradical activity than typical antioxidants such as resveratrol, ascorbic acid or Trolox in the lipid medium. The hydroperoxyl radical scavenging of SFR in the aqueous solution is approximately 530 times faster than that of Trolox and similar to ascorbic acid or resveratrol. This suggests that SFR is a promising radical scavenger in physiological environments.

Introduction

<pan class="Chemical">span class="Chemical">Sulfuretinpan> (SFR, figure 1) is a natural <sppan>an class="Chemical">flavonoid present in numerous plant species, including <span class="Species">Rhus verniciflua [1,2], Toxicodendron vernicifluum [3], Dahlia, Bidens tripartite and Dipterx lacunifera [4]. This compound is known to have numerous biological activities such as amelioration of rheumatoid arthritis symptoms [5], antimutagenic [6], antiplatelet [7], anti-cancer [8,9], anti-inflammatory effects [5,10], liver protection [11], anti-ageing effect for skin [12], anti-obesity effect [12] and antioxidant activity [2,13-15].

Figure 1

Molecular structure and atomic numbering of SFR.

Jung and co-workers [2] reported that SFR presented strong antioxidant activity in the <pan class="Chemical">span class="Chemical">DPPHpan> (<sppan>an class="Chemical">2,2-diphenyl-1-picrylhydrazyl) assay and total anti-<span class="Chemical">ROS (reactive oxygen species) activity with IC50 = 8.52 and 0.73 µM, respectively. The DPPH inhibition of SFR was about two times higher than that of L-ascorbic acid, whereas the total ROS inhibition is about five times stronger than Trolox. SFR also presented significant activity against ONOO− and HO• radicals [2]. Chen et al. [14] also reported that SFR has good DPPH, ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and HO• radical scavenging activity that is higher than butylated hydroxytoluene (BHT).

Although the antioxidant activity of SFR is broadly examined experimentally [2,14], there are no studies on the mechanism and kinetics of its antiradical activity, particularly in physiological environments. Computer calculations offer a convenient way to predict the antioxidant activity of organic compounds in physiological media [16-23]. In this context and as a continuation of our previous studies [18,24,25], we set out in this work to evaluate the <span class="Chemical">HOO• antiradical activity of SFR by a combination of thermodynamic and kinetic calculations. This study also considered the effects of solvents on the antioxidant properties of SFR in comparison with some typical antioxidants.

Computational details

All calculations were carried out with Gaussian 09 suite of programs [26]. M06–2X/6–311 + +G(d,p) model chemistry was used for all calculations [27-29]. It was demonstrated before that the M06–2X functional is one of the most reliable methods to study thermodynamics and kinetics of radical reactions, particularly in physiological environments [19,28,30,31]. The solvation model density (<span class="Disease">SMD) method was used for including the effects of <span class="Chemical">water and pentyl ethanoate in the computations [17,18,24,32-34]. The kinetic calculations were performed following the quantum mechanics-based test for the overall free radical scavenging activity (QM-ORSA) protocol [17,34], using the conventional transition state theory (TST) and 1 M standard state at 298.15 K [18,34-40]. The details of the method are shown in the electronic supplementary material, table S1.

Results and discussion

The HOO• antiradical activity of SFR in the gas phase

Thermodynamic evaluation

For SFR that contains OH and moieties, the antioxidant activity may follow either of the four main mechanisms: the formal <pan class="Chemical">span class="Chemical">hydrogenpan> transfer (FHT), the sequential proton loss electron transfer (SPLET), the single-electron transfer proton transfer (SETPT) and radical adduct formation (RAF) [41,42]. The first three pathways are defined by the following thermodynamic parameters: bond dissociation enthalpy (<sppan>an class="Chemical">BDE), proton affinity (PA) and ionization energy (IE), respectively. The Gibbs free energy change of the addition reaction is calculated directly for the RAF mechanism. Thus, the <span class="Chemical">BDE, IE and PA values of SFR were first calculated in the gas phase, and the results are shown in table 1.

Table 1

The calculated thermodynamic parameters (BDEs, PAs and IEs) of SFR in the gas phase.

positions	BDE	PA	IE
O6−H	90.7	323.4	174.6
O3′−H	80.5	327.9
O4′−H	77.5	320.9

The calculated thermodynamic parameters (<pan class="Chemical">span class="Chemical">BDEpan>s, PAs and IEs) of SFR in the gas phase.

As per table 1, the lowest <pan class="Chemical">span class="Chemical">BDEpan> value was predicted for O4′−H at 77.5 kcal mol−1. This value is lower than that of natural antioxidants such as <sppan>an class="Chemical">viniferifuran (82.7 kcal mol−1) [43], <span class="Chemical">resveratrol (83.9 kcal mol−1) [43], puerarin (87.3 kcal mol−1) [44] and vanillic acid (85.2 kcal mol−1) [45]. The lowest PA and IE values are about 4.14 and 2.25 times higher than the BDE value. Thus, based on the computed data, the antioxidant activity of SFR is predicted to favour the FHT pathway, at least in apolar and low-dielectric environments.

To confirm that FHT is indeed the preferred pathway of the <span class="Chemical">HOO• antiradical activity of SFR, the Gibbs free energy of the SFR + <span class="Chemical">HOO• reaction was calculated according to each of the four mechanisms: FHT, single-electron transfer (SET, the first step of the SETPT mechanism), sequential proton (SP, the first step of the SPLET) and RAF (table 2). It was found that the HOO• antiradical activity of SFR is only clearly spontaneous for FHT at O3′(O4′)−H bonds and RAF at the C8 position (ΔGo < 0), whereas the RAF reaction at C2 with ΔGo = 1.1 kcal mol−1 cannot be clearly excluded based on thermodynamics alone and therefore it was also included in the kinetic study. The other reactions are clearly not spontaneous with high positive ΔGo values. The ΔGo values for the reactions following the SP and SET pathways are much higher than those of the FHT mechanism. Thus, the calculated data suggest that the HOO• antiradical activity of SFR may follow either FHT or RAF mechanism (at O3′(4′)−H and C2/C8 positions, respectively), and these pathways should be investigated in the kinetic study.

Table 2

Calculated ΔGo (kcal/mol) of the SFR + HOO• reactions according to the FHT, SP, RAF and SET mechanisms in the gas phase.

positions	FHT	SP	SET	RAF
O6−H	4.8	170.8	152.1	—
O3′−H	−4.9	176.1		—
O4′−H	−7.7	169.2		—
C2	—	—		1.1
C8	—	—		−4.6

Calculated ΔGo (kcal/mol) of the SFR + <span class="Chemical">HOO• reactions according to the FHT, SP, RAF and SET mechanisms in the gas phase.

Kinetic study

Based on the above results, the kinetics of the SFR + <span class="Chemical">HOO• reaction in the gas phase was investigated for the thermodynamically favourable positions and mechanisms according to the QM-ORSA protocol [17], and the data are presented in table 3 and figure 2.

Table 3

Calculated ΔH (kcal/mol), activation Gibbs free energies (ΔG≠, kcal/mol), tunnelling corrections (κ), kEck (M−1 s−1) and branching ratios (Γ, %) for the HOO• + SFR reaction in the gas phase.

mechanism	positions	ΔH	ΔG≠	κ	kEck	Γ
FHT	O3′−H	2.3	11.6	39.6	8.43 × 105	23.0
	O4′−H	2.0	11.2	72.1	2.83 × 106	77.0
RAF	C2	7.1	17.1	1.5	2.83	0.0
	C8	8.6	17.7	1.5	9.03 × 10−1	0.0
koverall					3.67 × 106

Figure 2

The optimized transition state (TS) structures following the FHT and RAF mechanisms of the SFR + HOO• reaction (G: gas phase; W: water; P: pentyl ethanoate).

Calculated ΔH (kcal/mol), activation Gibbs free energies (ΔG≠, kcal/mol), <pan class="Chemical">span class="Disease">tunnellingpan> corrections (κ), kEck (M−1 s−1) and branching ratios (Γ, %) for the <sppan>an class="Chemical">HOO• + SFR reaction in the gas phase.

The optimized transition state (TS) structures following the FHT and RAF mechanisms of the SFR + <span class="Chemical">HOO• reaction (G: gas phase; W: <span class="Chemical">water; P: pentyl ethanoate).

It is apparent that the <span class="Chemical">HOO• antiradical activity of SFR occurs mostly by the H-abstraction of the O4′−H bond (ΔG≠ = 11.2 kcal/mol; kEck = 2.83 × 106 M−1 s−1; Γ = 77.0%). That is more than three times higher contribution than the <span class="Chemical">hydrogen abstraction of the O3′−H bond (ΔG≠ = 11.6 kcal mol−1; kEck = 8.43 × 105 M−1 s−1; Γ = 23.0%). By contrast, the addition of the radical does not make any contribution (Γ = 0%) at either the C2 or C8 positions. This result is in good agreement with previous studies in phenolic compounds [46-48]. We can conclude that the HOO• antiradical activity of SFR is dominated by the FHT mechanism at the O3′(4′)–H bond; therefore, this is further analysed in physiological environments.

The HOO• antiradical activity of SFR in physiological environments

Acid–base equilibrium

Previous studies showed that the deprotonation of the OH bonds plays a key role in the <span class="Chemical">HOO• antiradical activity of phenolic compounds in the aqueous solution [30,34,49]. The spontaneous dissociation of acidic moieties practically eliminates the activation energy barrier of the first step of the SPLET mechanism, simplifying it to direct electron transfer, and for this reason, this pathway can become energetically favoured in aqueous solution for the dissociated species. Thus, in this study, the deprotonation of SFR must also be considered. The PA values (table 1) showed that the site most likely to dissociate is the O4′−H bond. Thus, this bond was used to calculate the pKa of SFR. The pKa was computed following the literature [49,50], and the results are shown in figure 3. The calculated pKa value was 7.47. Thus, under physiologically relevant conditions (pH = 7.40), SFR has both neutral (HA, 54.0%) and anionic (A−, 46.0%) forms. Therefore, in the physiological environments, these states were used for the kinetic investigation.

Figure 3

The acid dissociation equilibrium of SFR.

Based on the results of the kinetic calculations in the gas phase, the <span class="Chemical">HOO• antiradical activity in non-polar environments was modelled by the <span class="Chemical">hydrogen transfer mechanism at the O3′(O4′)−H bonds. In the aqueous environment, the SET mechanism was also investigated for the deprotonated state of SFR. The overall rate constants (koverall) were computed following the QM-ORSA protocol [17,33], (table 4) according to equations (3.1) and (3.2).

Table 4

Calculated ΔG≠ (kcal mol−1), tunnelling corrections (κ), the nuclear reorganization energy (λ, kcal mol−1) rate constants (kapp, k, and koverall M−1 s−1), molar fractions (f) and branching ratios (Γ, %) at 298.15 K, in the SFR + HOO• reaction in pentyl ethanoate and water solvents.

mechanism		pentyl ethanoate				water
		ΔG≠	κ	kapp	Γ	ΔG≠	κ	kapp	f	kf**	Γ
SET						6.6	15.6*	8.90 × 107	0.460	4.09 × 107	86.2
HAT	O3′−H	15.0	106.9	6.90 × 103	38.5	16.0	744.5	9.20 × 103	0.540	4.97 × 103	0.0
	O4′−H	14.9	163.1	1.10 × 104	61.5	15.5	202.8	5.30 × 103	0.540	2.86 × 103	0.0
	O3′−H (anion)					7.8	1.2	1.42 × 107	0.460	6.53 × 106	13.7
koverall				1.79 × 104						4.75 × 107

*λ; **kf = f.kapp; Γ = k.100/koverall.

Calculated ΔG≠ (kcal mol−1), <pan class="Chemical">span class="Disease">tunnellingpan> corrections (κ), the nuclear reorganization energy (λ, kcal mol−1) rate constants (kapp, k, and koverall M−1 s−1), molar fractions (f) and branching ratios (Γ, %) at 298.15 K, in the SFR + <sppan>an class="Chemical">HOO• reaction in <span class="Chemical">pentyl ethanoate and water solvents.

In the <pan class="Chemical">span class="Chemical">lipidpan> medium

In <pan class="Chemical">span class="Chemical">waterpan> at pH = 7.40

As shown in table 4, the <span class="Chemical">HOO• antiradical activity of SFR in the polar solvent is excellent with the koverall = 4.75 × 107 M−1 s−1. Similarly, in the <span class="Chemical">lipid medium, SFR exhibits good activity with koverall = 1.79 × 104 M−1 s−1. It was found that the SET of anion A− plays a principal role (k = 4.09 × 107 M−1 s−1, Γ = 86.2%) in the radical scavenging activity of SFR. The H-abstraction of the anion state contributes about 13.7% to the overall rate constants. The rate constants for the H-abstraction of O3′(O4′)−H bonds against HOO• radical are k = 4.97 × 103 and 2.86 × 103 M−1 s−1, respectively; however, these reactions do not make any contributions (approx. 0%) to the activity of SFR. Based on the results, SFR is better HOO• radical scavenger than typical antioxidants Trolox, ascorbic acid and resveratrol in lipid phase (reference lipid phase activities: koverall = 3.40 × 103 M−1 s−1 [33], koverall = 5.71 × 103 M−1 s−1 [17] and koverall = 1.31 × 104 M−1 s−1 [46], respectively). In aqueous solution, the HOO• antiradical activity of SFR is approximately 530 times faster than that of Trolox (k = 8.96 × 104 M−1 s−1) [33] and fairly similar to other well-known natural antioxidants, i.e. ascorbic acid (k = 9.97 × 107 M−1 s−1) [17] and resveratrol (k = 5.62 × 107 M−1 s−1) [46]. Thus, the results suggest that SFR is a promising antioxidant in physiological media.

Conclusion

The <pan class="Chemical">span class="Chemical">hydroperoxyl radicalpan> scavenging activity of SFR was investigated using DFT calculations. The results showed that SFR has excellent <sppan>an class="Chemical">HOO• antiradical activity with koverall = 4.75 × 107 M−1 s−1 in <span class="Chemical">water at pH = 7.40 by the SET pathway of the anion state, and good/moderate HOO• scavenging activity in lipid environment (koverall = 1.79 × 104 M−1 s−1) by the FHT mechanism via the O3′(O4′)–H bonds. The hydroperoxyl antiradical activity of SFR is better than Trolox, ascorbic acid and resveratrol in the lipid medium. This activity of SFR is approximately 530 times faster than that of Trolox and relatively similar to ascorbic acid and resveratrol in the polar environment. Thus, SFR can be a useful natural antioxidant in physiological environments.

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